Sex determination in mammals is well understood at this time. In essentially all mammalian species, females have two XX chromosomes and males have one X and one Y chromosome. The important role of the Y chromosome as a determiner of male characteristics is apparent in view of the rare individuals with numerical abnormalities of the sex chromosomes. Individuals with two XX chromosomes and one y, Klinefelter's syndrome, are phenotypically male, while individuals with a single X and no Y, Turner's syndrome, are female. Similarly, XO mice and pigs are female, while XXY mice and cats are male.
The sex chromosomes in birds are the reverse of mammals. Male birds are homogametic, having two Z chromosomes, while females are heterogametic, having one Z and one W chromosome. As a result, the sex of the offspring is determined by the genetic content of the egg rather than the sperm.
Individual mammalian spermatozoa, being haploid, contain either a single X or a single Y chromosome, while the haploid eggs all contain single X chromosomes. The sperm therefore determines the sex of the conceptus. A single ejaculate contains many millions of sperm cells, with male and female determining types usually present in roughly equal amounts.
There are tremendous economic incentives in the livestock industry to develop a means for providing sperm for artificial insemination for which the ratio of one sex to the other sex is known. In the dairy industry, there is a greater demand for female offspring. In the pig industry in the United States, there is more preference for female is for pork production. Fractionations of semen by sex have been attempted based on a variety of physical, biochemical, and immunological methods. For the most part, successes have been marginal. A key part of any chemical or biochemical separation procedure is the assay of fractions. The assays now available, such as cytological examination of chromosomes or direct examination of live births, tend to be prohibitively tedious, slow, or expensive.
The best studied mammalian chromosomes are those of man, yet only a few genes thus far have been mapped on the human Y chromosome, possibly due to its small size. To the extent that the human gene map reflects the situation in farm animals, there are some Y-linked genes involved in fertility and growth that might be important in livestock production.
A prominent feature of the human Y chromosome DNA is a highly repeated sequence not found in females, as described by Bostock, et al, Nature 272,324-328 (1978) and Cooke et al, Chromosoma 87,491-502 (1982). Due to its repetitive nature, this material can be visualized directly as a 3.4 kilobase ethidium-staining band by agarose el electrophoresis of male DNA after digestion with the restriction enzyme HaeIII. This sequence occurs as a tandem array of several thousand copies, possibly representing as much as 30% of the human Y chromosome.
The existence of comparable Y-specific repeats in economically important livestock animals is not well-documented. See Epplen et al, Proc.Natl.Acad.Sci.USA 79,3798-3802 (1982); Phillips et al, Nature, 297,241-243 (1982); Ohno et al, Sexual Differentiation; Basic and Clinical Aspects, M. Serio et al. ed. (Raven Press, N.Y. 1984). Although there is cytological evidence indicating that the Y chromosome has been well-conserved in evolution among the various mammalian species, reported by Matthey and Vorontsov, Cytotaxonomy and Vertebrate Evolution. Chiarelli and Capanna, eds.,531-553 (Academic Press, London 1973), Research by Leonard et al, Therioenology 27,248 (Proc.Ann.Conf.Internat.Embryo Transfer Soc., Dublin, Ireland, Jan. 25-27, 1987) indicates that the human male specific HaeIII DNA fragment is not suitable as a sex-specific probe in cattle.
Leonard et al describe the use of a Y chromosome-specific DNA probe in the sexing of bovine embryos. Embryos were collected at 7-8 days of gestation from superovulated heifers. Biopsies consisting of 10-20 trophoblastic cells were assayed by in situ hybridization with a biotinylated Y-specific DNA probe. Male embryos were identified by their positive hybridization signals, as detected by an immunocytochemical procedure. This bovine sex-specific sequence consists of a 49 nucleotide tandem repetition which is cleavable by the restriction enzyme Sau3A. The same sequence is detectable as a seven kilobase EcoRI fragment in genomic Southern blots.
It is therefore an object of the present invention to provide a method for identifying sex-specific DNA's in livestock animals, especially pigs and chickens.
It is another object of the present invention to clone and characterize sex-specific DNA sequences.
It is a further object of the present invention to provide sex-specific DNA probes.
It is a still further object to provide methods and probes to assay the sex ratios in semen fractions or unfertilized eggs.
It is another object of the present invention to provide a method and probes to determine the sex of embryos.